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Rosmarinic acid derivatives from Salvia

officinalis

Article in Phytochemistry · May 1999

DOI: 10.1016/S0031-9422(98)00730-4

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 \
PERGAMON Phytochemistry 40 "0888# 80Ð83

Rosmarinic acid derivatives from Salvia of_cinalis

Yinrong Lu\ L[ Yeap Foo
Industrial Research Limited\ P[O[ Box 20209\ Lower Hutt\ New Zealand

Received 17 October 0887

Abstract

Sagerinic acid\ a novel cyclobutane and salvianolic acid K\ derived from rosmarinic acid\ were isolated together with the parent
compound from polar solvent extracts of Salvia of_cinalis[ Their chemical structures were elucidated by NMR and\ for sagerinic
acid\ the stereochemistry of the substituents on the cyclobutane moiety was established as 2b\3a!diaryl!0a\1b!dicarboxylic acid
diester "m!truxinate form#[ Þ 0888 Elsevier Science Ltd[ All rights reserved[

Keywords] Salvia of_cinalis^ Labiatae^ Rosmarinic acid^ Salvianolic acid K^ Sagerinic acid^ m!truxinate

0[ Introduction 1[ Results and discussion

Sage "Salvia# is a popular herb which has been used in a
variety of food preparations since ancient times[ Judging
from its Latin name\ sage has clearly long enjoyed a
reputation in folklore for its health giving properties and
for treating all kinds of ailments "Keller\ 0867#[ Although
today the most widespread use of the herb is in ~avouring
foods\ it is still commonly used as a household remedy\
mainly as an aid in drying up the ~ow of mother|s milk\
in reducing saliva secretion\ as an anhidrotic to control
night sweats associated with illness and in relieving oral
cavity and throat in~ammations "Steinegger + Hansel\
0877#[ Sage has been observed to have excellent proper!
ties in inhibiting lipid peroxidation and this activity is
attributed principally to the presence of phenolic com!
pounds\ such as carnosic acid\ carnosol and rosmarinic
acid "Zhang\ Bao\ Wu\ Rosen\ + Ho\ 0889^ Cuvelier\
Berset\ + Richard\ 0883^ Cuvelier\ Richard\ + Berset\
0885#[ Our interest in sage was prompted by its history
of health properties and enhanced by recent studies "Ai
+ Li\ 0877^ Tanaka et al[\ 0878^ Tanaka\ Nishimura\
Kouno\ Nonaka\ + Young\ 0885^ Tanaka\ Nishimura\
Kouno\ Nonaka\ + Yang\ 0886#\ which revealed the pres!
ence of some unusual compounds that could be recovered
after the volatile ~avouring ingredients had been
removed[
 Corresponding author[
Column chromatography of sage extract on Sephadex
LH19 yielded rosmarinic acid "0# as the main compound[
Its identity was evident from its 0H and 02C NMR spectra
which were consistent with published data "Kelly\
Harru}\ + Carmack\ 0865^ Eicher\ Ott\ + Speicher\
0885#[ Also isolated were two other compounds\ 1 and 2[
The 0H NMR spectrum of compound 1 showed two
doublets at d 6[21 and 5[03 which on the basis of the
observed large proton!proton coupling "J 05[9 Hz# were
assigned to a pair of trans!ole_nic protons[ In addition\
there were three ABX!spin systems "Table 0# observed in
the aromatic region\ which were assignable to the three
discrete sets of protons of the 2\3!dihydroxyphenyl unit[
Also observed were two sets of doublets at d 3[69 and
4[98 "J4[0 Hz# in the low _eld region\ suggesting two
neighbouring methine protons attached to oxygen!bear!
ing carbons and a further three sets of multiplets at d
2[99\ 2[03 and 4[94 attributable to three protons coupled
in an ABX pattern "J03[1\ 8[0 and 2[7 Hz#\ consistent
with the presence of a ÐCH"OH#ÐCH1Ð unit[ The 02C
NMR spectrum of 1 "Table 1# showed the presence of
three carbonyl carbons\ of which two were identi_able
with those of carboxylic acids "d 068[8 and 067[5# and
one of a carboxyl ester "d 060[3#[ The presence of three
sets of 2\3!dihydroxyphenyl groups was con_rmed by the
appearance of 07 aromatic carbons consisting of nine
quaternary carbons\ of which six were phenoxyl carbons
"d 035Ð040# and nine tertiary carbons "d 006Ð014#[ In
addition\ there were also two ole_nic carbons "d 037[1

9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S 9 9 2 0 ! 8 3 1 1 " 8 7 # 9 9 6 2 9 ! 3
81 Y[ Lu\ L[Y[ Foo : Phytochemistry 40 "0888# 80Ð83
and 007[9#\ three oxygenated methine carbons "d 75[6\
68[1 and 65[3# and one methylene carbon "d 28[5# present
in the spectrum[ These chemical shifts\ corroborated by
0
H!0H and 0H!02C COSY studies\ were consistent with
the presence of a rosmarinic acid and a b!"2\3!dihy!
droxyphenyl#glyceric acid moieties in the structure of 1[
The manner in which the rosmarinic acid and b!"2\3!
dihydroxyphenyl#glyceric acid were linked together in 1
was established by application of a long!range coupling
experiment "HMBC# which showed coupling between the
H!7ý "d 3[6# and C!3 "d 040[0#\ hence\ the ether linkage
was at the a!OH of glyceric acid and the C"3#!OH of the
ca}eoyl moiety in rosmarinic acid[ The full assignments
of 0H and 02C chemical shifts of 1 are shown in Tables 0Ð
1\ respectively[ Its chemical structure was also con_rmed
by electrospray mass spectroscopy "ESMS# which
showed an ðMÐHŁ− at m:z 444 and a base peak at m:z
165[7 corresponding to ðMÐ1HŁ1− due to double ioni!
zation[ Compound 1 was therefore identical to salvianolic
acid K which was recently reported to be present in the
roots of Chinese desert sage "Salvia deserta# "Tezuka et
al[\ 0887#[
The 02C NMR spectrum of sagerinic acid "2# "Table 1#
showed some similarity to that of rosmarinic acid\ except
that there were twice as many carbon signals present as
those observed for rosmarinic acid[ The eight phenoxyl
carbon signals "d 034Ð035#\ 01 tertiary carbon signals "d
006Ð013# and four quaternary carbon signals "d 021Ð
022# "established using DEPT# were consistent with the
presence of four sets of 2\3!dihydroxyphenyl groups[ This
was corroborated by the 0H NMR spectrum "Table 0#
which showed 01 aromatic protons "d 5[4Ð6[0# coupled
in manner consistent with four discrete ABX!resonance
systems[ The 0H NMR spectrum also showed couplings
between the protons at d 1[4Ð1[8 and those at d 3[2Ð3[3\
these protons being associated with the corresponding
four aliphatic carbons at d 26[26\ 27[88\ 68[37 and 68[66\
respectively\ as shown by HMQC\ thus indicating the
presence of two ÐCH"OH#ÐCH1Ð moieties[ Sagerinic acid
di}ered from rosmarinic acid in the absence of the ole_nic
 Y[ Lu\ L[Y[ Foo : Phytochemistry 40 "0888# 80Ð83 82

Table 0 Table 1
0 02
H NMR "299 MHz\ D1O# data for rosmarinic acid "0#\ salvianolic acid C NMR "64 MHz\ D1O# data for rosmarinic acid "0#\ salvianolic acid
K "1# and sagerinic acid "2# K "1# and sagerinic acid "2#

No[ 0a 1 2 Carbon 0a 1 2

1 6[06 d "0[8# 5[80 s 5[45 d "1[0# 0 018[4 020[1 022[5

4 5[77 d "7[7# 5[56 d "7[3# 5[47 d "7[1# 1 006[2 007[9 007[4
5 6[99 dd "7[7\ 1[9# 5[64 d "7:3# 4[88 dd "7[1\ 1[0# 2 032[3 037[5 035[0
6 6[42 d "05[9# 6[21 d "05[9# 3[96 dd "09[5\ 5[4# 3 034[5 040[0 034[3
7 5[23 d "05[9# 5[03 d "05[9# 2[59 dd "09[5\ 5[4# 4 007[4 007[0 007[7
1? 5[78 br s 5[80 s 5[69 d "1[9# 5 013[0 013[7 019[8
4? 5[64 d "7[9# 5[76 d "7[0# 5[61 d "7[0# 6 037[6 037[1 32[6
5? 5[60 dd "7[9\ 0[6# 5[68 d "7[0# 5[48 dd "7[0\ 1[9# 7 006[9 007[9 49[0
6? 1[84 dd "02[7\ 7[7# 2[99 dd "03[1\ 8[0# 1[60 dd "03[9\ 8[6# 8 060[3 060[3 065[4
2[05 d "02[7# 2[03 dd "03[1\ 2[7# 1[74 dd "03[9\ 2[4# 0? 022[0 022[9 021[6
7? 4[96 d "7[7# 4[94 dd "8[0\ 2[7# 3[24 dd "8[2\ 2[6# 1? 008[5 008[8 008[4
1ý 6[96 s 5[62 d "1[1# 2? 035[7 035[4 035[4
4ý 5[78 d "7[2# 5[52 d "7[1# 3? 036[4 034[2 034[2
5ý 5[81 d "7[2# 5[27 dd "7[2\ 1[0# 4? 007[6 007[5 007[5
6ý 4[98 d "4[0# 3[97 dd "09[5\ 6[7# 5? 014[1 013[6 013[5
7ý 3[69 d "4[0# 2[69 dd "09[5\ 6[7# 6? 28[8 28[5 28[3
11 5[59 d "1[9# 7? 68[2 68[1 68[7
41 5[69 d "7[0# 8? 068[4 068[8 068[1
51 5[30 dd "7[0\ 1[9# 0ý 023[7 022[7
61 1[41 dd "03[2\ 3[7#^ 1ý 006[8 006[5
1[51 dd "03[2\ 6[5# 2ý 035[4 035[1
71 3[22 dd "6[5\ 3[7# 3ý 035[3 034[5
4ý 007[8 007[6
a
Measured in CD2OD[ 5ý 011[6 011[7
6ý 65[3 32[9
7ý 75[6 37[8
8ý 067[5 064[8
carbons\ which were replaced by four tertiary aliphatic 01 021[2
carbons at d 32[92\ 32[60\ 37[80 and 49[09 "established 11 008[6
by DEPT#[ The methine protons at d 2[5Ð2[6 and 3[9Ð 21 035[3
3[0\ associated with these carbons were mutually coupled 31 034[0
to one another and\ therefore\ were covalently linked 41 007[6
51 013[2
in a cyclobutane ring[ Thus\ sagerinic acid was a novel 61 28[9
rosmarinic acid dimer\ where dimerization had occurred 71 68[4
by a ð1¦1Ł union of the ole_nic moieties[ The cyclobutane 81 068[0
structure of sagerinic acid was further supported by a
Measured in CD2OD[
ESMS using negative ion detection\ which gave a promi!
nent parent ion at m:z 608 consistent with ðMÐHŁ− and
a base peak at m:z 248 corresponding to ðMÐ1HŁ1−[
The complete assignment of the 0H and 02C NMR
cinnamic acid amides are also known to be present in the
chemical shift data of sagerinic acid was achieved by
cell walls of tropical grasses "Ford + Hartley\ 0889^
extensive use of 0HÐ0H COSY\ 0HÐ02C COSY and inverse
Morrison\ Robertson\ Stewart\ + Wightman\ 0880#\
long range 0H\ 02C coupling "HMBC# experiments[ The
con_guration of sagerinic acid was established using bamboos "Tachibana\ Ohkubo\ + Towers\ 0881a\ 0881b#
NOESY which showed positive interactions between H! and Piper species "Filho\ De Souza\ + Mattos\ 0870^
5 "d 4[88# and H!6ý "d 3[97#\ H!5 and H!7ý "d 2[69#\ as Duh\ Wu\ + Wang\ 0889^ Maxwell + Rampersad\ 0880#[
well as those between H!5ý "d 5[27# and H!6 "d 3[96#\ H! More recently\ stachysetin\ an analogous apigenin!6!p!
5ý and H!7 "d 2[59#[ Similar NOESY e}ects were also coumaroylglucoside cyclodimer in the m!truxinate form\
observed between protons H!5 "d 4[88# and H!7? "d 3[24#\ was reported in the aerial parts of Stachys aegyptiaca
H!7? and H!7ý "d 2[69#\ and H!7 "d\ 2[59# and H!71 "d "El!Ansari\ Nawwar\ + Saleh\ 0884#[
3[22#[ These clearly indicated that the substitutents on
the cyclobutane ring was in a m!truxinate arrangement 2[ Experimental
"Green + Rejto\ 0863#[ Sagerinic acid is possibly formed
0
from rosmarinic acid via a photochemical cyclization H and 02C NMR were recorded on a Bruker AC 299
process[ Similar dimeric products with cyclobutane struc! instrument and chemical shifts "d# were referenced to the
tures derived from p!coumaric acid\ ferulic acid and solvent signal[ HPLC was performed on a LiChrospherþ
83 Y[ Lu\ L[Y[ Foo : Phytochemistry 40 "0888# 80Ð83
099 RP!07 "4 mm# column "014×3 mm# held at 29>C with
the following solvent program] solvent A\ 1) HOAc in
H1O^ solvent B\ 1) HOAc in MeCN^ starting from 3 up
to 01) B in 19 min\ to 19) B in 29 min and to 49) B
in 34 min[ The ~ow rate was 0 ml min−0 and detection
was made at 179 nm[
2[0[ Extraction\ fractionation and isolation
The residue after supercritical CO1 extraction of
ground S[ of_cinalis "49 g# was extracted with 69) aq[
Me1CO "2×499 ml# and the combined extracts conc[ and
freeze!dried to yield 00 g of solid material "11)#[ The
extract "09 g# was fractionated on a HP19 column "19×5
cm# into a H1O fr[ "499 ml# and a MeOH fr[ "299 ml#\
which were conc[ and freeze!dried to a}ord 3[4 g "H1O
fr[# and 3[5 g "MeOH fr[#\ respectively[ These fr[ were
separately chromatographed on a Sephadex LH19
column "49×1[4 cm# and eluted with aq[ MeOH "up to
29)#[ Frs[ were collected and monitored by HPLC and
the sorted frs[ conc and freeze!dried[ Where necessary
frs[ were further puri_ed on Sephadex LH19 until
chromatographically pure compounds were obtained[
2[1[ Rosmarinic acid "0#
1429 mg from MeOH fr[ HPLC Rt 29[7 min[ 0H and
02
C NMR] Tables 0Ð1[
2[2[ Salvianolic acid K "1#
177 mg from H1O fr[ HPLC Rt 20[5 min[ ESÐMS
"negative mode# m:z "rel[ int[# 444 "ðMÐHŁ− 2#\ 166 "ðMÐ
1HŁ1− 099#[ UV lmax
MeOH
nm "log o#\ 101 "3[11#\ 177 "3[04#\
211 sh "2[86#[ H and 02C NMR] Tables 0Ð1[
0 Tanaka\ T[\ Morimoto\ S[\ Nonaka\ G[\ Nishioka\ I[\ Yokozawa\ T[\
2[3[ Sagerinic acid "2#
54 mg from H1O fr[ HPLC Rt 18[3 min[ ðaŁD19 ¦3>
"MeOH c 9[1#[ ESÐMS "negative mode# m:z "rel[ int[#
608[2 "ðMÐHŁ− 2#\ 248[1 "ðMÐ1HŁ1− 099#[ UV lmax
MeOH
nm
0 02
"log o# 121 "3[97#\ 175 "3[98#[ H and C NMR] Tables
0Ð1[
View publication stats
Acknowledgements
The authors thank Dr[ Herbert Wong for NMR spec!
tra and Mrs[ Wendy Jackson of the University of
Waikato for ESÐMS data[
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